Synthesis and in vitro characterization of a P2X7 radioligand [I]TZ6019 and its response to neuroinflammation in a mouse model of Alzheimer disease.

The purinergic receptor P2X ligand-gated ion channel 7 (P2X7 receptor) is a promising imaging target to detect neuroinflammation. Herein, we report development of a potent iodinated radiotracer for P2X7 receptor, [I]TZ6019. The radiosynthesis of [I]TZ6019 was accomplished by allylic-tin precursor iodination using [I]NaI with good radiochemical yield of 85% and high radiochemical purity of > 99%. Human embryonic kidney 293 (HEK-293) cell line stably transfected with the human P2X7 receptor was used to characterize the binding affinity of TZ6019 by fluorescence, radioactive competitive, and saturation binding assays. A radioligand competitive binding assay with [I]TZ6019 demonstrated that the nonradioactive compound TZ6019 has an IC value of 9.49 ± 1.4nM, and the known P2X7 receptor compound GSK1482160 has an IC value of 4.30 ± 0.86nM, consistent with previous reports. The radioligand saturation binding assay and competitive assay revealed that [I]TZ6019 specifically bound to the human P2X7 receptor with high affinity (K = 6.3 ± 0.9nM). In vitro autoradiography quantification with brain slices collected from 9-month old P301S tau transgenic mice along with wild type controls, revealed higher binding of [I]TZ6019 (35% increase) in the brain of P301S transgenic mice (n = 3, p = 0.04) compared to wild type controls. The immunofluorescence microscopy confirmed that expression of P2X7 receptor was colocalized with astrocytes in the tauopathy P301S transgenic mice. [I]TZ6019 has specific binding for P2X7 receptor and has great potential to be a radiotracer for screening new compounds and quantifying expression of P2X7 receptor in neuroinflammation related diseases.